Aryl Hydrocarbon Receptor Transcriptional Activity Research Articles

AIP mutations impair AhR signaling in pituitary adenoma patients fibroblasts and in GH3 cells.

Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene predispose humans to pituitary adenomas through unknown molecular mechanisms. The best-known interacting partner of AIP is the aryl hydrocarbon receptor (AhR), a transcription factor that mediates the effects of xenobiotics implicated in carcinogenesis. As 75% of AIP mutations disrupt the physical and/or functional interaction with AhR, we postulated that the tumorigenic potential of AIP mutations might result from altered AhR signaling. We evaluated the impact of AIP mutations on the AhR signaling pathway, first in fibroblasts from AIP-mutated patients with pituitary adenomas, by comparison with fibroblasts from healthy subjects, then in transfected pituitary GH3 cells. The AIP protein level in mutated fibroblasts was about half of that in cells from healthy subjects, but AhR expression was unaffected. Gene expression analyses showed significant modifications in the expression of the AhR target genes CYP1B1 and AHRR in AIP-mutated fibroblasts, both before and after stimulation with the endogenous AhR ligand kynurenine. Kynurenine increased Cyp1b1 expression to a greater extent in GH3 cells overexpressing wild type compared with cells expressing mutant AIP Knockdown of endogenous Aip in these cells attenuated Cyp1b1 induction by the AhR ligand. Both mutant AIP expression and knockdown of endogenous Aip affected the kynurenine-dependent GH secretion of GH3 cells. This study of human fibroblasts bearing endogenous heterozygous AIP mutations and transfected pituitary GH3 cells shows that AIP mutations affect the AIP protein level and alter AhR transcriptional activity in a gene- and tissue-dependent manner.

AHR promoter variant modulates its transcription and downstream effectors by allele-specific AHR-SP1 interaction functioning as a genetic marker for vitiligo.

Vitiligo is an acquired depigmentation disorder largely caused by defective melanocyte- or autoimmunity-induced melanocyte destruction. The aryl hydrocarbon receptor (AHR) is essential for melanocyte homeostasis and immune process, and abnormal AHR was observed in vitiligo. We previously identified the T allele of AHR −129C > T variant as a protective factor against vitiligo. However, biological characterization underlying such effects is not fully certain, further validation by mechanistic research is warranted and was conducted in the present study. We showed that −129T allele promoted AHR transcriptional activity through facilitating its interaction with SP1 transcription factor (SP1) compared with −129C allele. We subsequently found reduced peripheral AHR and SP1 transcript expressions in vitiligo and a negative correlation of AHR level with disease duration. We also investigated AHR-related cytokines and observed increased serum TNF-α concentration and diminished serum levels of IL-10 and TGF-β1 in vitiligo. Further genetic analysis showed that -129T carriers possessed higher levels of AHR and IL-10 than −129C carriers. Therefore, our study indicates that the modulation of AHR transcription by a promoter variant has a profound influence on vitiligo, not only advancing our understanding on AHR function but also providing novel insight into the pathogenesis of degenerative or autoimmune diseases including vitiligo.

Optical Isomers of Atorvastatin, Rosuvastatin and Fluvastatin Enantiospecifically Activate Pregnane X Receptor PXR and Induce CYP2A6, CYP2B6 and CYP3A4 in Human Hepatocytes.

Atorvastatin, fluvastatin and rosuvastatin are drugs used for treatment of hypercholesterolemia. They cause numerous drug-drug interactions by inhibiting and inducing drug-metabolizing cytochromes P450. These three statins exist in four optical forms, but they are currently used as enantiopure drugs, i.e., only one single enantiomer. There are numerous evidences that efficacy, adverse effects and toxicity of drugs may be enantiospecific. Therefore, we investigated the effects of optical isomers of atorvastatin, fluvastatin and rosuvastatin on the expression of drug-metabolizing P450s in primary human hepatocytes, using western blots and RT-PCR for measurement of proteins and mRNAs, respectively. The activity of P450 transcriptional regulators, including pregnane X receptor (PXR), aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR), was assessed by gene reporter assays and EMSA. Transcriptional activity of AhR was not influenced by any statin tested. Basal transcriptional activity of GR was not affected by tested statins, but dexamethasone-inducible activity of GR was dose-dependently and enantioselectively inhibited by fluvastatin. Basal and ligand-inducible transcriptional activity of PXR was dose-dependently influenced by all tested statins, and the potency and efficacy between individual optical isomers varied depending on statin and optical isomer. The expression of CYP1A1 and CYP1A2 in human hepatocytes was not influenced by tested statins. All statins induced CYP2A6, CYP2B6 and CYP3A4, and the effects on CYP2C9 were rather modulatory. The effects varied between statins and enantiomers and induction potency decreased in order: atorvastatin (RR>RS = SR>SS) > fluvastatin (SR>RS = SS>RR) >> rosuvastatin (only RS active). The data presented here might be of toxicological and clinical importance.

Agonistic effect of selected isoflavones on arylhydrocarbon receptor in a novel AZ-AhR transgenic gene reporter human cell line.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that controls the expression of a diverse set of genes. Structurally diverse compounds bind to AhR and act as AhR agonists. Well characterised family of natural AhR ligands are isoflavones, which are compounds found predominantly in soy beans or red clover. In this study we have examined agonistic effect of selected isoflavones (genistein, daidzein, biochanin A, formononetin and equol) on AhR in the novel transgenic gene reporter human cell line AZ-AhR, a stably transfected AhR-responsive cell line allowing rapid and sensitive assessment of AhR transcriptional activity. We demonstrated that biochanin A, formononetin and genistein at concentration 10(-4) mol/l exerted agonistic effects on AhR with fold activation of 309- fold, 108-fold and 27-fold, which is about 84.8%, 29.6% and 7.4%, respectively, of the value attained by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Daidzein and equol did not show any significant effects on AhR.

L-kynurenine/aryl hydrocarbon receptor pathway mediates brain damage after experimental stroke.

Aryl hydrocarbon receptor (AhR) is a transcription factor that belongs to the basic helix-loop-helix PAS (Per-Arnt-Sim homology domain) family known to mediate the toxic and carcinogenic effects of xenobiotics. Interestingly, AhR is widely expressed in the central nervous system, but its physiological and pathological roles are still unclear. To define the role of AhR in stroke, we used middle cerebral artery occlusion in mice and oxygen-glucose deprivation in rat cortical neurons. The results presented here show that the ischemic insult increases total and nuclear AhR levels and AhR transcriptional activity in neurons in vivo and in vitro. We also show that AhR has a causal role in acute ischemic damage because pharmacological or genetic loss-of-function approaches result in neuroprotection. Inhibition of cAMP response element-binding protein-dependent signaling may participate in the deleterious actions of AhR. Finally, we have also found that L-kynurenine, a tryptophan metabolite with AhR agonistic properties, is an endogenous ligand that mediates AhR activation in the brain after middle cerebral artery occlusion. Our data demonstrate that an L-kynurenine/AhR pathway mediates acute brain damage after stroke and open new possibilities for the diagnosis and treatment of this pathology.

Hypoxia perturbs aryl hydrocarbon receptor signaling and CYP1A1 expression induced by PCB 126 in human skin and liver-derived cell lines

The aryl hydrocarbon receptor (AhR) is an important mediator of toxic responses after exposure to xenobiotics including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like polychlorinated biphenyls (PCBs). Activation of AhR responsive genes requires AhR dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT), a heterodimeric partner also shared by the hypoxia-inducible factor-1α (HIF-1α) protein. TCDD-stimulated AhR transcriptional activity can be influenced by hypoxia; however, it less well known whether hypoxia interferes with AhR transcriptional transactivation in the context of PCB-mediated AhR activation in human cells. Elucidation of this interaction is important in liver hepatocytes which extensively metabolize ingested PCBs and experience varying degrees of oxygen tension during normal physiologic function. This study was designed to assess the effect of hypoxia on AhR transcriptional responses after exposure to 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126). Exposure to 1% O2 prior to PCB 126 treatment significantly inhibited CYP1A1 mRNA and protein expression in human HepG2 and HaCaT cells. CYP1A1 transcriptional activation was significantly decreased upon PCB 126 stimulation under conditions of hypoxia. Additionally, hypoxia pre-treatment reduced PCB 126 induced AhR binding to CYP1 target gene promoters. Importantly, ARNT overexpression rescued cells from the inhibitory effect of hypoxia on XRE-luciferase reporter activity. Therefore, the mechanism of interference of the signaling crosstalk between the AhR and hypoxia pathways appears to be at least in part dependent on ARNT availability. Our results show that AhR activation and CYP1A1 expression induced by PCB 126 were significantly inhibited by hypoxia and hypoxia might therefore play an important role in PCB metabolism and toxicity.

RelB and the aryl hydrocarbon receptor: dendritic cell tolerance at the epithelial interface

RelB and the aryl hydrocarbon receptor: dendritic cell tolerance at the epithelial interface

Novel cell line for assessment of aryl hydrocarbon receptor transcriptional activity

Novel cell line for assessment of aryl hydrocarbon receptor transcriptional activity

Regulation of the Activity and Expression of Aryl Hydrocarbon Receptor by Ethanol in Mouse Hepatic Stellate Cells

During the course of alcohol-induced liver damage, hepatic stellate cells are transformed into proliferative, fibrogenic, and contractile myofibroblasts. Aryl hydrocarbon receptor (AhR) is a transcription factor that controls the expression of genes involved in the metabolism of xenobiotics, inflammation, cell proliferation, and death. Immortal mouse hepatic stellate cells (MHSCs) were isolated from transgenic mice that expressed a thermolabile SV40 tumor antigen. Quantitative real-time reverse transcription polymerase chain reaction assays, Western blot analysis, promoter activity assays, and chromatin immunoprecipitation analyses were performed for studying the effect of ethanol (EtOH) on AhR expression and transcriptional activity. Treatment of MHSCs with 50 to 200mM EtOH for 6hours induced AhR nuclear translocation, enhanced the promoter activity of cytochrome P450 (CYP) 1A1, increased the amount of AhR bound to the promoter of CYP1A1 and 1B1, and up-regulated the mRNA expression of these AhR target genes in a dose-dependent manner. In contrast, EtOH exposure down-regulated AhR mRNA and protein expression. Similarly, benzo(a)pyrene (BaP) at 10nM reduced AhR and increased CYP1A1 and 1B1 mRNAs. Pretreatment of MHSCs with 50mM EtOH for 7days diminished the capacity of MHSCs to express CYP1A1 and 1B1 induced by a 200mM EtOH challenge, or by 10nM BaP. However, the up-regulatory effect of EtOH on solute carrier family 16, member 6 (SLC16a6) was unaffected by EtOH pretreatment. Similar to EtOH, dimethyl sulfoxide (DMSO) at concentrations of 50 to 100mM down-regulated AhR and up-regulated CYP1A1 mRNA expression in a dose-dependent manner. These data, for the first time, demonstrate that EtOH activates MHSC AhR and down-regulates its expression. Chronic EtOH pretreatment lowers the availability of AhR, and specifically diminishes the inducibility of CYP genes. The effect on AhR appears to not be an EtOH-specific response, as DMSO alone (and possibly other organic solvents) was also able to activate AhR.

Aryl hydrocarbon receptor activation by aminoflavone: New molecular target for renal cancer treatment

Aminoflavone (AF; NSC 686288, AFP464, NSC710464) is a new anticancer drug that has recently entered phase II clinical trials. It has demonstrated antiproliferative effects in MCF-7 human breast cancer cells mediated by the aryl hydrocarbon receptor (AhR). AF also exhibits noteworthy evidence of antitumor activity in vitro and in vivo against neoplastic cells of renal origin. AF treatment of sensitive renal cells, in contrast to resistant cells, promotes the induction of CYP1A1, the covalent binding of AF-reactive intermediates and apoptosis. Based on this evidence, the aim of this study was to evaluate the role of AhR, the main transcriptional regulator of CYP1A1, in the antiproliferative effects of AF in human renal cancer cells. AF-cytoxicity in human renal cell lines and a renal cancer cell strain was assessed by MTS assay in the presence or absence of an Ahr inhibitor. Drug-induced AhR nuclear translocation was evaluated by western blotting of AhR in cytosolic and nuclear fractions and by measuring xenobiotic response element-driven luciferase activity. Apoptosis induced by the drug was evaluated by 4,6-diamidino-2-phenylindole and acridine orange/ethidium bromide staining and by measuring phosphorylated P53 (p-P53) and P21 levels, caspase 3 activation and poly(ADP-ribose) polymerase cleavage. AF inhibited cell growth in a dose-dependent manner in TK-10, Caki-1, SN12-C and A498 human renal cells but not in ACHN cells. The antiproliferative effect of AF was abrogated by pre-incubation of TK-10, Caki-1 and SN12-C cells with the AhR antagonist, α-naphthoflavone. AF treatment also induced apoptosis in TK-10, Caki-1 and SN12-C cells, which was not observed in ACHN cells. AF induced time-dependent AhR nuclear translocation and AhR transcriptional activity in sensitive renal cancer cell lines. A renal cell strain derived from a human papillary tumor also showed sensitivity to AF, as well as AhR pathway activation and drug-induced apoptosis. AhR translocation could be included as a marker of sensitivity to AF in sensitive renal tumor cells of different histological origin, in ongoing phase II clinical trials.

Novel Stably Transfected Gene Reporter Human Hepatoma Cell Line for Assessment of Aryl Hydrocarbon Receptor Transcriptional Activity: Construction and Characterization

We constructed stably transfected gene reporter cell line AZ-AHR, allowing measurement of aryl hydrocarbon receptor (AhR) transcriptional activity. Human hepatoma HepG2 cells were transfected with a construct containing several AhR binding sites upstream of luciferase reporter gene. We prepared 12 clones and we characterized the best five in responsiveness to TCDD. Dose-response analyses were performed for various AhR ligands, including TCDD, 3-methylcholanthrene, indirubin, resveratrol, omeprazole, and SP600125. The EC(50) values were similar in all tested clones. Induction of luciferase was time-dependent, and treatment for 6 h with 5 nM TCDD was sufficient to evaluate AhR transcriptional activity in 96-well plate format (8-24 fold induction). Response to AhR ligands of cryopreserved cells after thawing was not significantly different from that of fresh cells. Cell line remained fully responsive to AhR ligands over 15 passages and 30 days in culture without significant alterations. Overall, we have developed novel human luciferase reporter cell line AZ-AHR for monitoring AhR transcriptional activity. The sensitivity of the assay allows high throughput format (96-well plate) and evaluation of luciferase activity as soon as after 6 h of incubation, which has potential implication for studies of cytotoxic compounds.

Hes1 Expression Impacts Megakaryocytic Polyploidization and May Contribute to the Platelet Defects Found In Aryl Hydrocarbon Receptor (AhR)-Null Mice.

Abstract 2612 Microarray analyses (n = 3) from our lab uncovered that Aryl hydrocarbon receptor (AhR) mRNA increased 4–6 fold in Megakaryocytes (Mks) compared to isogenic granulocytic cultures and identified the Aryl hydrocarbon receptor as a novel transcriptional activator of megakaryocytic differentiation and maturation (Lindsey et al. Blood, 2009). (Q)-RT-PCR and Western Blot analyses confirmed these findings in differentiating Mks derived from primary human CD34+ cells and differentiating CHRF cells (human megakaryoblastic leukemia cell line). Unpublished data from our lab found that AhR-null mice (Schmidt et al. Proc Natl Acad Sci, 1996) exhibited a 9% decrease in platelet counts compared to WT mice. There were also 10.4% fewer reticulated young, RNA-containing platelets in AhR-null mice compared to WT mice, indicating a defect in either platelet generation or Mk maturation. We examined platelet functionality and found that while the average bleeding time for WT mice was approximately 1.5 minutes, AhR-null mice bled on average 8 minutes, or roughly 5.3 times longer. We also calculated the volume of blood loss and found that AhR-null mice lost 3 times as much blood during these assays as WT mice. To investigate if these platelet defects were due to abnormal Mk maturation, we examined the level of steady-state polyploidization of Mks residing within the murine bone marrow niche and found that compared to WT mice, AhR-null mice had ca. 25% fewer high ploidy (≥ 32n) Mks, but more Mks in the lower ploidy classes of 8n and 16n. Together, these data suggest that AhR-null mice exhibit platelet defects of both number and function, and steady-state Mks from AhR-null mice are less polyploid than WT mice. Previous EMSA experiments (n = 4) had found that AhR transcriptional activity, measured by the binding of AhR to a consensus DNA binding site, increased during Mk polyploidization and that decreased Mk ploidy correlated to a 2.7 fold decrease in expression of Hes1, an AhR target gene (n = 3; p= 0.018). Therefore, we hypothesized that AhR impacts Mk ploidy through transcriptional regulation of Hes1. In our current work, EMSA experiments (n = 3) showed a 3.5 fold increase of AhR binding to oligonucleotides corresponding to a putative AhR binding site within the Hes1 promoter, indicating that AhR could transcriptionaly regulate Hes1 during megakaryopoiesis. As an important component and regulator of the Notch signaling cascade, Hes1 regulates the cell cycle and cellular quiescence (Sang et al. Science, 2008), but its role during normal Mk differentiation remains unexplored. Interestingly, mRNA expression of Dlk1, a Hes1 transcriptional target, is increased in CD34+ cells isolated from MDS patients compared to CD34+ cells from normal individuals (Sakajiri et al. Leukemia, 2005), suggesting that Hes1 target genes may be deregulated in megakaryoblastic leukemia. To determine if Hes1 could be involved in Mk differentiation, we performed (Q)-RT-PCR experiments on differentiating Mks, granulocytes, and erythrocytes ex vivo expanded from primary human CD34+ cells. We found that by day 12 Hes1 expression increases 2.8 fold in Mks, but decreases more than 2.9 fold in isogenic granulocytes (n = 2 for each; p = 0.044). Hes1 expression appears to remain unchanged during erythroid development (n = 2). To investigate the functional impact of Hes1 during megakaryopoiesis, we used RNAi-mediated knockdown of Hes1 to generate two independent KD-Hes1 cell lines. These cell lines express 68 and 59 percent less Hes1 mRNA (n = 3; p = 0.008 for Hes1-A and p = 0.015 for Hes1-B), 31 and 47 percent less Hes1 protein (n = 3), without impacting AhR expression (n = 3; p = 0.46). Similar to knocking down AhR, Mk-ploidy distributions in KD-Hes1 cells were shifted towards lower ploidy classes, and were incapable of reaching higher ploidy classes (i.e., ≥ 32n) seen in control cells. Although there was little impact in undifferentiated CHRF cells, ploidy levels on day 7 (maximal ploidy in control cells) were between 25.3 and 26.7 percent less than control cells depending on the cell line (n = 3; p < 0.05 for both cell lines). These experiments genetically link Hes1 and AhR and suggest that AhR regulation of Hes1 during megakaryopoiesis mediates megakaryocytic polyploidization. As such, we provide a novel molecular model of endomitotic entry and Mk polyploidization, while also providing a mechanistic explanation for the platelet defects found in AhR null mice. Disclosures: No relevant conflicts of interest to declare.

Abstract 4364: Mechanism of promotion effect of diphenylarsenic acid on rat liver carcinogenesis

Abstract Diphenylarsinic acid (DPAA) is an enviromental degradation of product of diphenylarsine chloride or diphenylarsine cyanide, which were chemical warfare agents produced by Japan during the World War II. DPAA is now considered a dangerous environmental pollution in Kamisu, Japan, where it is suspected of inducing health effect that include articulation disorders, involuntary movements, and sleep disorders. We previously showed that GST-P (Glutathione S-transferase P) positive foci which is precancerous condition increased the number and the area at DPAA 20 ppm but not at below on rat liver carcinogenesis in medium-term bioassay (Ito test), suggesting that DPAA had promotion activities. However, the mechanism of its promotion activities is unclear. The purpose of present study is to clarify the mechanism of DPAA-induced promotion activity in rat liver carcinogenesis. We found that DPAA at 20 ppm but not at below increased significantly in oxidative DNA damage, mRNA expression of CYP1B1, cyclin D1, and c-Myc, and protein level and transcriptional activity of AhR (Arylhydrocarbon receptor) which is implicated in most of the biological responses to the environmental contaminant (e.g. TCDD). Proteome analysis showed that 69 proteins were differentially up or down-regulated in DEN-DPAA 20ppm group compared with DEN alone including 18 proteins of AhR pathway. These results suggested that activity of AhR pathway and oxidative DNA damage on rat liver carcinogenesis involved in promotion activity of DPAA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4364.

Abstract 4453: Aryl hydrocarbon receptor activation by Aminoflavone: New molecular target for renal cancer treatment

Abstract Aminoflavone (4H-1-benzopyran-4-one, 5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methyl; AF; NSC 686288) is a new anticancer drug that recently entered Phase I clinical trials. Previous work of our laboratory has demonstrated that the antiproliferative effect of AF in MCF-7 human breast cancer cells is mediated by the Aryl hydrocarbon Receptor (AhR). Treatment of MCF-7 cells with AF resulted in AhR nuclear translocation generating an increase in AhR-target genes expression, such as CYP1A1. It has been demonstrated that CYP1A1 induction is necessary for the conversion of AF pro-drug into several metabolites that cause cell apoptosis. AF also exhibits noteworthy evidence of antitumor activity in vitro against neoplastic cells of renal origin. In vivo, the drug produced tumor-free, long term survivors in mice bearing subcutaneous renal xenografts. These studies performed on renal cell lines and renal cell carcinoma isolates obtained from patients’ tumors suggest that renal papillary tumors were generally more sensitive to AF than the clear cell tumors. AF treatment of sensitive renal cells, in contrast to resistant cells, promotes CYP1A1 induction, covalent binding of AF-reactive intermediates to several macromolecules and apoptosis. Based on this evidence, the aim of this study was to evaluate the role of AhR pathway in the antiproliferative effect of AF in human renal cancer cells. Our results show that AF (0.1-1 μM) inhibited cell growth evaluated by MTS assay in a dose dependent manner in TK10 (47 ± 5% of control, p<0.01) and SN12-c (47 ± 6% of control, p<0.01) human renal cells but not in ACHN cells (96 ± 4% of control). AF treatment (1 μM, 72 h) also induced apoptosis in TK10 and SN12c cells but not in ACHN cells. In TK10 and SN12-c sensitive cell lines AF (1 μM) induced time dependent AhR nuclear translocation evaluated by Western blot and immunofluorescence. This effect was not seen in resistant ACHN cells. In addition, AF induced AhR transcriptional activity shown by induction of XRE-Luciferase in TK10 (AF 10 μM p<0,01 vs control) and SN12-c (AF 0,1 μM p<0,05 vs control) sensitive cells but not in ACHN cells. Finally, the antiproliferative effect of AF was abrogated by preincubation of TK10 (80 ± 16%, p<0.01 vs AF) and SN12-c (63 ± 6%, p<0.01 vs AF) cells with the AhR antagonist, α-napthoflavone (αNF, 1 μM). In other experiments, a different AhR antagonist, 4,7-phenanthroline (10 μM), partially reversed growth inhibition by AF in TK10 cells. These data indicate that AhR-mediated signal transduction is necessary for the cytotoxic effect of AF in sensitive renal tumor cells. Therefore, AhR translocation could be included as a marker of sensitivity to AF in future phase II trials. AF acting through the AhR pathway is a promising new targeted treatment for renal cancer cases that are refractory to other therapeutic agents currently used in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4453.

Modeling of the Aryl Hydrocarbon Receptor (AhR) Ligand Binding Domain and Its Utility in Virtual Ligand Screening to Predict New AhR Ligands

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor; the AhR Per-AhR/Arnt-Sim (PAS) domain binds ligands. We developed homology models of the AhR PAS domain to characterize previously observed intra- and interspecies differences in ligand binding using molecular docking. In silico structure-based virtual ligand screening using our model resulted in the identification of pinocembrin and 5-hydroxy-7-methoxyflavone, which promoted nuclear translocation and transcriptional activation of AhR and AhR-dependent induction of endogenous target genes.

Repression of Aryl Hydrocarbon Receptor Transcriptional Activity by Epidermal Growth Factor

The aryl hydrocarbon receptor (AHR) mediates most, if not all, of the many toxicological effects of the environmental pollutant 2,3,7,8-tetrachlorodibenzo- p-dioxin [(TCDD) or dioxin]. The "classical" pathway of AHR action involves dimerization of the liganded AHR with the aryl hydrocarbon nuclear translocator (ARNT) protein, and the AHR-ARNT dimer specifically associates with the enhancer regions of dioxin-responsive genes, leading to their increased transcription. Sutter and coworkers recently reported that epidermal growth factor (EGF) represses the dioxin-mediated induction of CYP1A1 in cultured normal human keratinocytes by inhibiting the recruitment of the transcriptional coactivator protein p300 to the CYP1A1 gene. EGF also inhibits the dioxin-dependent induction of certain parameters in keratinocytes that are reflective of dioxin-induced chloracne. These findings point to the potential usefulness of EGF for the treatment of chloracne and also describe a novel mechanism for repression of dioxin-induced gene transcription.

Microarray analysis of genes induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) in thymic progenitor cell populations prior to thymic atrophy (84.3)

Abstract TCDD activates the Aryl Hydrocarbon Receptor (AhR), a basic helix-loop-helix transcription factor, and causes thymic atrophy in all species examined. We have previously shown [Laiosa, et. al. J. Immunol. (2004) 171 4582-91] that the earliest detectable effect associated with TCDD induced thymic atrophy was a decline in the percentage of S phase cells in the CD3-CD4-CD8- triple negative (TN) intrathymic progenitor 3 and 4 stages within 9-12 hours of treatment, prior to any detectable reductions in thymocyte cell number. We also demonstrated that this effect was strictly by activation of the AhR in the developing lymphocytes. We now show, using microarray analysis, that 108 distinct genes are affected in the TN1-4 thymic progenitor stages 6 and 12 hours after TCDD treatment. While most of these genes have specific AhR recognition sequences, only 7 genes were altered exclusively in the TN3 and 4 stages. Interestingly, all 7 of these genes demonstrated a decrease in expression. These 7 genes represent novel targets of AhR transcriptional activity in the developing immune system. Future work will test the hypothesis that these 7 genes play a critical role for thymocyte cell cycle regulation and proliferation following TCRβ selection. Funded by 5RO1- ES04862 (TAG) and Hendricks Fund (Syracuse University).

Modulation of aryl hydrocarbon receptor activity by four and a half LIM domain 2

The aryl hydrocarbon receptor (AhR) mediates transcriptional effects of a diverse array of ligands including environmental contaminants that have been linked to various cancers. The transcriptional activity of the AhR is modulated by different coregulators such as the p160 family members of coactivators and nuclear receptor coactivator 4 (NcoA4). In this study, we provide novel evidence that four and a half LIM only protein 2 (FHL2) interacts with and differentially modulates the transcriptional activity of AhR. Co-immunoprecipitation studies indicate that FHL2 interacts with AhR in a ligand-independent manner but not with its heterodimeric partner, AhR nuclear translocator (ARNT). Overexpression of FHL2 enhanced AhR-mediated expression of a luciferase reporter gene in a dose- and ligand-dependent manner in COS cells. Furthermore, FHL2 cooperated with NcoA4 to synergistically enhance AhR transcriptional activity in these cells. However, the impact of FHL2 on AhR transcriptional activity was cell-specific: FHL2 facilitated AhR action in MCF-7 and PC-3 cells, whereas it suppressed AhR activity in T47D and LNCaP cells. These results of reporter gene studies were corroborated by the impact of FHL2 overexpression on, an established target gene of AhR, cytochrome P450 (CYP1A1) expression. We also demonstrated a potential competition of AhR and androgen receptor (AR) for FHL2 availability in COS cells, as FHL2-facilitation was significantly decreased in the presence of liganded AR. These findings indicate a functional interaction between AhR and FHL2 that modulates the activity of AhR and therefore could affect its role in cancer progression or development.

Leukotriene A4 Metabolites Are Endogenous Ligands for the Ah Receptor

In addition to orchestrating an adaptive metabolic response to xenobiotic compounds, the aryl hydrocarbon receptor (AHR) also plays a necessary role in the normal physiology of mice. The AHR is activated by a structurally diverse group of chemicals ranging from carcinogenic environmental pollutants to dietary metabolites and a number of endogenous molecules. Leukotriene A 4 (5,6-LTA 4) metabolites were identified in DRE-driven luciferase reporter assays as activators of AHR signaling. Various LTA 4 metabolites, including several 5,6- and 5,12-DiHETE products, were screened for AHR activity with 6- trans-LTB 4, 6- trans-12- epi-LTB 4, 5( S),6( S)-DiHETE, and 5( S),6( R)-DiHETE eliciting a significant level of AHR transcriptional activity. However, electrophoretic mobility shift assays (EMSAs) revealed that only 5,6-DiHETE isomers were capable of directly binding and activating the AHR to a DNA-binding species in vitro. Furthermore, ligand competition binding experiments confirm the ability of these compounds to directly bind to the AHR. Interestingly, "aged" preparations of 5,6-DiHETE isomers produced an enhanced level of AHR activation while demonstrating an increase in binding affinity for the receptor. Although the reason for this has not been fully determined, the formation of geometric isomers in the conjugated triene region of these molecules may play a role in the observed increase in AHR-mediated transcriptional activity. This work suggests a connection between AHR activation and inflammatory signaling molecules produced by the 5-lipoxygenase pathway.

Inflammatory Signaling and Aryl Hydrocarbon Receptor Mediate Synergistic Induction of Interleukin 6 in MCF-7 Cells

The pleiotropic cytokine interleukin 6 (IL-6) is involved in immune cell homeostasis. Additionally, IL-6 expression and signaling in tumor cells have been shown to elicit both protumor and antitumor properties. There is a plethora of mechanistic knowledge regarding how IL-6 signal transduction translates to biological responses. However, there is little understanding as to what factors control IL-6 expression within a tumor cell environment. The studies presented herein show that, in MCF-7 breast and ECC-1 endocervical cancer cells, the stimulation of aryl hydrocarbon receptor (AHR) activity, in combination with IL-1beta or phorbol 12-myristate 13-acetate (PMA) treatment, results in a marked synergistic induction of IL-6 levels over what is seen without AHR activation. Chromatin immunoprecipitation experiments suggest that the regulation of IL-6 mRNA expression occurs at the chromatin level, as AHR presence on the IL-6 promoter was observed in response to treatment with AHR ligand. Synergistic induction of IL-6 expression was sustained for 72 hours, with accumulation of IL-6 protein reaching levels 4.8-fold above IL-1beta treatment alone. In addition, transcriptional regulation of the prototypic AHR responsive gene Cyp1a1 was negatively regulated by PMA and IL-1beta treatment. Silencing of RELA expression alleviated IL-1beta-mediated repression of AHR transcriptional activity, whereas PMA-mediated repression was maintained. Additionally, small interfering RNA studies reveal that AHR and RELA are necessary for synergistic induction of IL-6. The findings presented here reveal the AHR as a potential therapeutic target for selective modulation of IL-6 expression in some tumor cell types. The data also suggest a possible previously unrecognized mechanism of AHR-mediated tumor promotion.